The project described here involves the investigation of new approaches to improve capillary electrophoresis (CE) methodology for the biochemical and clinical laboratories. One of the barriers to the implementation of CE methodology, either in capillary or microchip form, is poor concentration sensitivity when conventional UV absorbance detection is employed. This project will investigate the use of both rapid polarity switching and reaction product stacking with etectrophoretically mediated microanalysis (EMMA), with an eye towards enhancing the sensitivity of clinical applications of this relatively new methodology. The project will be in three phases: The initial experiments will be carried out using the well-known Jaffe method, a widely used methodology for the determination of creatinine in clinical biofluids, as a model system. The Jaffe method involves the reaction of creatinine with alkaline picrate. Preliminary experiments have indicated that the use of rapid polarity switching during the on-column mixing step of an EMMA analysis improves the sensitivity of the method. In addition, since the product of the on-column reaction is doubly anionic, it should be possible to evoke transient isotachophoretic (tlTP) stacking of the product to further enhance the analytical signal (phase 2). The third phase of this project will involve the application of the EMMA methodology with rapid polarity switching -- and stacking, if possible -- to other chemical systems including kinetically challenged (i.e. relatively slow) reactions involving small molecules as well as enzymatic reactions. In all, this project will elucidate the capability of rapid polarity switching to enhance product formation when employed with EMMA, and may lead to new approaches for rapid, low-volume bioassays in the clinical laboratory.
